THE MECHANISM OF LUTEINIZATION IN VITRO AND IN VIVO

将继续研究颗粒细胞黄体化的机制 重点放在本地控制过程上。卵泡的作用 大(6-12 mm)猪分离的液体黄体刺激因子(LS) 将检查卵泡。LS是一种大约60,000的蛋白质 道尔顿病毒将从大的猪卵泡液中分离出来，纯化并 具有化学和生理特征的。这个LS将被化验 其增强促黄体生成素/人绒毛膜促性腺激素受体和促性腺激素释放激素能力的基础 猪颗粒细胞培养中孕酮的分泌。LS将会是 通过适当的酒精沉淀组合进行提纯， Sephacryl-S-300柱层析、DEAE离子交换层析 在TSK 5 PW色谱柱上进行三丙烯基和高效液相色谱离子交换。C3反相液相色谱 将携带相柱和TIYO苏打3000西南凝胶排斥柱 按要求退出。级分将用十二烷基硫酸钠凝胶进行表征 电泳法和蛋白水解酶测定法。最终产品将以 以结束组分析和手动埃德曼降级。它的蜂窝位置 纯化的LS在培养的猪颗粒中的作用将被检测 细胞，即其改变黄体生成素/人绒毛膜促性腺激素受体从头合成的能力 利用重氨基酸的掺入，改变腺苷环化酶的能力， &改变一般蛋白质合成的能力。大鼠颗粒细胞将 也可作为实验系统使用。一种抗血清、单克隆和 将产生针对猪LS的多克隆。交叉反应性 将检测人类卵泡液中的抗LS活性。这个 猪颗粒细胞和膜细胞分泌LS的能力 在不同的荷尔蒙条件下进行检查。LS在体内的作用 将首先在未成熟的大鼠身上进行检测，如果阳性结果是 获得的观察结果将在未成熟的恒河猴身上进行。 猴子。体内LS抗血清对卵巢的交替作用 功能将在未成熟的老鼠或猴子身上进行检测。一个人的角色 小猪卵泡液黄体生成抑制物(LI)的存在 将确定卵泡(1-3 mm)和中等大小(3-5 mm)的卵泡。 LI将用FSH处理的猪颗粒进行分离和纯化 细胞培养作为一种生物测定。抑制卵泡刺激素诱导的能力 黄体生成素/人绒毛膜促性腺激素受体、孕酮分泌与形态黄体生成 都会被追随。将制备一种针对Li的抗血清并进行评估 在体外和体内对李嘉诚的作用。荷尔蒙的控制 对颗粒细胞合成的LI活性进行检测。的交互作用 LI和LS在控制卵泡成熟方面将被考虑。

Studies on the mechanism of granulosa cell luteinization will be pursued with an emphasis on local control processes. The role of a follicular fluid luteinization stimulator (LS) isolated from large (6-12mm) porcine follicles will be examined. The LS, which is a protein about 60,000 daltons, will be isolated from large porcine follicular fluid, purified and characterized chemically and physiologically. This LS will be assayed on the basis of its ability to enhance FSH induction of LH/hCG receptors and progesterone secretion in cultured porcine granulosa cells. The LS will be purified by an appropriate combination of alcohol precipitation, Sephacryl-S-300 column chromatography, ion exchange chromatography on DEAE trisacryl and HPLC ion exchange on a TSK 5 PW column. HPLC on a C3 reverse phase column and a tiyo soda 3000 SW gel exclusion column will be carried out as required. Fractions will be characterized by SDS gel electrophoresis and by protease assay. The final product will be subject to end group analysis and Manual Edman degradation. The cellular site of action of the purified LS will be examined in cultured porcine granulosa cells, i.e. its ability to alter de novo synthesis of LH/hCG receptors using incorporation of heavy amino acids, ability to alter adenyl cyclase, & ability to alter general protein synthesis. Rat granulosa cells will also be used as an experimental system. An antiserum, monoclonal and polyclonal, will be generated against porcine LS. The cross reactivity against LS activity in human follicular fluid will be examined. The ability of porcine granulosa and thecal cells to secrete LS will be examined under different hormonal conditions. The effect of LS in vivo will be examined initially in the immature rat and if positive results are obtained the observations will be carried out in the immature rhesus monkey. Alternately the effects of antiserum to LS in vivo to ovarian function will be examined in the immature rat or monkey. The role of a follicular fluid luteinization inhibitor (LI) present in small porcine follicles (1-3mm) and medium (3-5mm) sized follicles will be determined. The LI will be isolated and purified using FSH treated porcine granulosa cell cultures as a bioassay. The ability to inhibit FSH induction of LH/hCG receptors, progesterone secretion and morphological luteinization will be followed. An antiserum against LI will be prepared and evaluated for actions against LI in vitro and in vivo. The hormonal control of granulosa cell synthesis of LI activity will be examined. Interaction of LI and LS in control of follicular maturation will be considered.